User:Greg Black/Sandbox 1

The anti-cancer drug, cisplatin
The discovery of the anti-cancer activity of cisplatin ([Pt(NH3)2Cl2] was serendipitous.



Discovery
Barnett Rosenberg, a physicist, thought that the picture of a cell dividing looked much like the pattern made by a dipole field with iron filings and a bar magnet, and he decided to investigate the cell division of the bacterium E. coli in the presence of a magnetic field. He found that cell division stopped but that the bacteria kept on growing, forming into long strands ref here. This effect had been caused by cisplatin, formed by the ammonium chloride nutrient, NH4Cl, reacting with the platinum electrodes under the effect of light and the electric field. The prevention of cell division is of course essential to cancer therapy. The next experiment was to inject doses of cisplatin into tumours in mice, and compare results against a control group: the tumours decreased dramatically in size. Medical research started immediately.

Dr Eve Wiltshore very rapidly showed that the drug had activity in ovarian cancer in particular and alongside that other trials showed that the drug was active in men with testicular cancer. Before the advent of cisplatin, in the case of men with testicular cancer the cure rate was something like 1 in 10; when cisplatin containing regimes came into clinical practice the cure rate went up to something like 9 in 10.

Cisplatin has side-effects, apart from nausea it can also cause kidney damage. It didn’t work against all cancers, and most of all there was a huge gap in understanding: how did it work, and why didn’t the trans- form of the same chemical have the same effect?

NMR proved to be an immensely useful tool in helping to determine both how cisplatin gets into the cells, and what happened to it once it was inside ref here. Cisplatin hydrolyses stepwise to produce positively charged ions. First, one chloride ion is replaced by water to form a species with a single positive charge and then the second chloride ion is replaced by a water molecule to an ion with two positive charges.

Test scene: cisplatin in the groove

pics here

When the Platinum complex becomes positively charged there would be a natural attraction for it to migrate to and bind to DNA which is a poly-anion. Cisplatin blocks the action of DNA - both replication and transcription.

Early NMR work ref here showed that most of the cisplatin formed what is called an adduct by linking to two neighbouring guanine bases on the same strand of the DNA through the N7 atom to form an intrastrand adduct.

pic here

More unusually, a very small amount of the cisplatin – less then 5% – bonded to the DNA so that the molecule actually straddled the two strands, making what is known as an interstrand adduct.

A major breakthrough came in the mid 1980s when Suzanne Sherman was successful in crystallizing the smallest building block on DNA bound to cisplatin ref here, and in 1995 Lippard’s group determined the crystal structure for the double stranded DNA complex that we see here.

Cisplatin is found to bind in the larger major groove. The platinum is still essentially square planar although the guanines are no longer parallel. The DNA molecule undergoes a significant bend, ranging from 50 to 80 degrees. Also the minor groove of the DNA substantially widens from the normal value in so-called B or classical B-form DNA of about 5 to 6 Ǻ up to 10 or 11 Ǻ. So opposite the cisplatin was a widened, flattened, minor groove. It seems to be this altered structure of DNA that leads to the recognition of other factors in the cell which ultimately produce the anti-cancer activity.

But there are also interstrand crosslinks that cross the two strands of DNA of which are maybe only 2 or so percent but there are a group of people who believe that they are the more important in terms of biological significance.

Proteins are the key to processes such as the repair mechanism. Normally, damaged DNA is recognised and marked for repair, which is done by cutting out the damage, so called excision repair. The bent platinated DNA seems to be recognised by other proteins – high mobility group, or HMG domains – which bind at the damaged site and prevent access by the repair proteins, protecting the adduct from excision repair. If this happened more efficiently in a tumour cell than in a normal cell of the same tissue that would be a wonderful mechanism for anti-cancer activity, and we're working hard to evaluate that hypothesis.

This structure shows an HMG protein – the red tube, attached to a natural binding site for transcription. It binds to the minor groove, but the similarity of the effect on the minor groove to the effect of cisplatin is striking. In both cases the minor groove becomes wider and shallower. Perhaps it is possible that the cisplatin DNA confuses the repair mechanism by this similarity and so escapes excision.

pic here – has this been proved?

Carboplatin
The problem with cisplatin was one of chemical reactivity – the rate at which those chloride ligands leave the molecule. And the whole idea behind carboplatin was to tone down that reactivity and in fact what is present in carboplatin instead of the chlorides is a cyclobutane dicarboxyato group which laboratory experiments have shown slows down the rate of aquation, the weight, the, the rate at which those ligands leave by about 10 fold in carboplatin and that has had a dramatic clinical effect in patients in that the kidney toxicity seen with cisplatin is almost totally absent with carboplatin and indeed the nausea and vomiting seen with cisplatin which is also a severe problem, is much less with carboplatin as well ref here.